Download Impaired overload-induced muscle growth is associated with

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J Physiol 574.1 (2006) pp 291–305
Impaired overload-induced muscle growth is associated
with diminished translational signalling in aged rat
fast-twitch skeletal muscle
David M. Thomson and Scott E. Gordon
Human Performance Laboratory, Department of Exercise and Sport Science, and Department of Physiology, East Carolina University,
Greenville, NC 27858, USA
Impaired overload-induced protein synthesis and growth in aged fast-twitch skeletal muscle
may result from diminished responsiveness of signalling intermediates controlling protein
translation. Yet, potential age-related signalling decrements have never been examined in
direct parallel with impaired overload-induced muscle growth in any model. To this end, we
used Western blotting to examine the contents and phosphorylation states of mammalian
target of rapamycin (mTOR) and its downstream translational signalling intermediates, 70 kDa
ribosomal protein S6 kinase (S6k), ribosomal protein S6 (rpS6), eukaryotic elongation factor 2
(eEF2), and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), in conjunction with
impaired growth in 1 week overloaded fast-twitch plantaris muscles (via unilateral gastrocnemius
ablation) of old (O; 30 months) versus young adult (YA; 8 months) male Fischer344 × Brown
Norway rats. The significantly (P ≤ 0.05) diminished growth (assessed by total muscle protein
content) in overloaded O muscles (5.6 ± 1.7 versus 19.3 ± 2.9% in YA) was accompanied by
significant impairments in the phosphorylation states of mTOR (Ser2448 ), S6k (impaired at the
mTOR-specific Thr389 residue but not at Thr421 /Ser424 ), rpS6 (Ser235/236 ) and 4E-BP1 (gel shift), as
well as deficits in total eEF2 accretion. Moreover, in overloaded muscles across both age groups,
phospho-S6k at Thr389 (but not at Thr421 /Ser424 ), 4E-BP1 phosphorylation status, and total
eEF2 accretion were all positively correlated with percentage muscle hypertrophy, and negatively
correlated with the phosphorylation (Thr172 ) of 5 -AMP-activated protein kinase (AMPK; which
inhibits translational signalling and protein synthesis in young muscle at rest). As previously
published by ourselves, AMPK was hyperphosphorylated in O versus YA muscles used in the
current investigation. The present results provide solid evidence that impaired overload-induced
growth in aged fast-twitch muscle may partly result from multiple-level decrements in signalling
pathway(s) controlling protein translation, and also provide an initial indication that AMPK
hyperactivation with age may potentially lie upstream of these decrements.
(Received 10 February 2006; accepted after revision 17 April 2006; first published online 20 April 2006)
Correspondingauthor S. E. Gordon: Human Performance Laboratory, 363 Ward Sports Medicine Building, East Carolina
University, Greenville, NC 27858, USA. Email: [email protected]
It is well known that ageing is accompanied by a loss
of muscle mass (sarcopenia), particularly in fast-twitch
fibres, a phenomenon which contributes to decreased
muscular strength and power (Welle, 2002). This is an
important problem for the elderly because it may lead to
an increased risk of injury, a decrease in general mobility
and loss of functional independence. While it is clear that
older individuals are capable of some muscle growth with
training, mechanical overload-induced muscle growth,
particularly of fast-twitch fibres, is attenuated in old
humans (Welle et al. 1996b; Häkkinen et al. 1998, 2001)
and rodents (Blough & Linderman, 2000; Alway et al.
C 2006 The Authors. Journal compilation C 2006 The Physiological Society
2002; Degens & Alway, 2003; Thomson & Gordon, 2005)
when compared with the young. However, the mechanisms
underlying this decreased capacity for fast-twitch skeletal
muscle growth in the aged are still poorly understood.
For muscle growth to occur, the rate of protein synthesis
must exceed that of protein breakdown. Protein synthesis
is regulated in part by the activity of initiation and
elongation factors that control the rate of mRNA translation. One critical signalling pathway controlling protein
synthesis during mechanically induced skeletal muscle
growth involves mammalian target of rapamycin (mTOR)
(Bodine et al. 2001; Nader & Esser, 2001; Reynolds et al.
DOI: 10.1113/jphysiol.2006.107490
292
D. M. Thomson and S. E. Gordon
2002). The mTOR protein lies downstream of Akt in
the insulin signalling pathway, but can also be regulated
through a variety of other hormonal, nutritional and
metabolic signals. When active, mTOR promotes an
increase in the efficiency and capacity for translation
of mRNA to protein by increasing, either directly or
indirectly, the activity of several proteins, including 70 kDa
ribosomal protein S6 kinase (S6k), ribosomal protein S6
(rpS6), eIF4E-binding protein 1 (4E-BP1), and eukaryotic
elongation factor 2 (eEF2) (Kimball et al. 1998; Nader et al.
2002). S6k promotes the translation of various ribosomal
proteins and elongation factors (including eEF2), presumably via phosphorylation of rpS6 (Terada et al. 1994;
Jefferies et al. 1997), and also promotes a general increase in
protein elongation by an indirect activation of eEF2 (Wang
et al. 2001). On the other hand, mTOR phosphorylation of
4E-BP1 promotes translation initiation of 5 -cap mRNAs,
including most eukaryotic mRNAs (Gingras et al. 1999;
Shah et al. 2000). With various forms of increased muscle
loading, mTOR and its downstream targets are activated
(Baar & Esser, 1999; Bodine et al. 2001; Nader & Esser, 2001;
Reynolds et al. 2002; Bolster et al. 2003b; Parkington et al.
2003) and muscle protein synthesis is enhanced (Wong &
Booth, 1990a,b; Hernandez et al. 2000; Pehme et al. 2004a).
Resting skeletal muscle protein synthesis declines with
age in most cases (Yarasheski et al. 1993; Welle et al.
1995), in part due to decreased translational efficiency
(Welle et al. 1996a). Mechanically induced increases in
protein synthesis, especially in fast-twitch muscle, are also
potentially attenuated with old age and may underlie
the diminished growth (Welle et al. 1995; Tamaki et al.
2000; Pehme et al. 2004b). Potential signalling deficits
underlying these age-related impairments are not well
characterized. Despite equivocal mTOR and S6k data in
resting muscle (Dardevet et al. 2000; Li et al. 2003; Guillet
et al. 2004; Kimball et al. 2004; Cuthbertson et al. 2005), the
responsiveness of S6k to various hormonal or nutritional
stimuli is clearly impaired in aged skeletal muscle
(Dardevet et al. 2000; Li et al. 2003; Guillet et al. 2004;
Cuthbertson et al. 2005). Less is known about age-related
decrements in mTOR, S6k and potential downstream
signalling with respect to loading stimuli; however, S6k
activation is diminished in association with failed regrowth
in aged slow-twitch muscle that has been reloaded after
disuse (Morris et al. 2004). Although there are reports
that mTOR and S6k responsiveness may potentially be
decreased with age after acute high-frequency electrical
stimulation in fast-twitch muscle (Parkington et al. 2003,
2004), the responses between age groups were not directly
compared in those studies. Furthermore, it is unknown
whether such signalling alterations with age corresponded
with impaired protein synthesis or impaired potential
for longer-term muscle growth in that model. Thus, the
potential connection between diminished mTOR and/or
S6k activation in aged fast-twitch muscle under conditions
J Physiol 574.1
known to demonstrate defective overload-induced protein
synthesis and growth remains completely unexplored,
especially with respect to downstream signalling intermediates (such as rpS6, eEF2 and 4E-BP1) controlling
protein translation and accretion. We postulated that
diminished mTOR, S6k, rpS6, eEF2 and 4E-BP1 signalling
would exist in response to compensatory overload in
aged fast-twitch muscle, a model demonstrating greatly
impaired muscle protein synthesis (Pehme et al. 2004b)
and growth (Thomson & Gordon, 2005) in aged animals.
Also unclear are the mechanism(s) of upstream
regulation of mTOR and other translational signalling
intermediates in aged and/or overloaded skeletal muscle.
In young resting skeletal muscle (Bolster et al. 2002) and
cardiac muscle (Horman et al. 2002; Chan et al. 2004),
protein synthesis is negatively regulated by activation
of the cellular energy sensor 5 -AMP-activated protein
kinase (AMPK). This effect is likely to be due, in part, to
AMPK’s inhibition of mTOR, S6k, 4E-BP1 and/or eEF2
(Bolster et al. 2002; Horman et al. 2002; Chan et al.
2004) signalling. Importantly, we have demonstrated that
AMPK phosphorylation is elevated with age in resting
and overloaded fast-twitch skeletal muscle (Thomson &
Gordon, 2005). Moreover, we found that the degree of
hypertrophy is negatively correlated with the amount of
AMPK phosphorylation in overloaded fast-twitch muscle
after synergist ablation (Thomson & Gordon, 2005). Thus,
we postulated that any potential impairments in mTOR,
S6k, rpS6, eEF2 and 4E-BP1 signalling would correspond
with the AMPK hyperphosphorylation and diminished
overload-induced growth we observed in aged fast-twitch
skeletal muscle (Thomson & Gordon, 2005). The purpose
of this investigation was therefore to examine the contents
and phosphorylation states of these proteins in response
to overload (via gastrocnemius ablation) in young adult
and old fast-twitch plantaris muscles.
Methods
Animals
Young adult (YA; 8 months; n = 7) and old (O; 30 months;
n = 7) Fischer344 × Brown Norway F1 hybrid (FBN)
male rats were housed in the animal care facility of
East Carolina University Brody School of Medicine. Body
weights, muscle weights and AMPK results have been previously reported for these animals (Thomson & Gordon,
2005). The FBN rat was chosen for use in this study
because it is considered to be the preferred model for the
study of age-related skeletal muscle dysfunction (Blough
& Linderman, 2000; Degens & Alway, 2003). The animals
were kept on a 12 h light–dark cycle and had free access
to water and chow. The East Carolina University Animal
Care and Use Committee approved all procedures prior to
experimentation.
C 2006 The Authors. Journal compilation C 2006 The Physiological Society
J Physiol 574.1
Overload and impaired translational signalling in aged skeletal muscle
Synergist ablation procedure
Unilateral 1 week overload of the plantaris muscle (93%
fast-twitch muscle by mass; Armstrong & Phelps, 1984)
was achieved through the surgical ablation of the gastrocnemius muscle. The 1 week overload period was chosen
because it is known that muscle protein synthesis in young
rats is elevated at this time point of compensatory overload (Baillie & Garlick, 1991), and continues to be elevated
at least 30 days after initiation of the stimulus (Pehme
et al. 2004a,b). Furthermore, rat plantaris muscle protein
synthesis in the compensatory overload model is greatly
diminished with age (Pehme et al. 2004b), and we have
shown that protein accretion is diminished by 71% in
aged rats during 1 week of overload in plantaris muscles
after gastrocnemius ablation (Thomson & Gordon, 2005).
The advantage of unilateral ablation over bilateral ablation
is that it allows within-animal comparisons to be
made between sham-operated (control) and overloaded
muscles, thus eliminating bias due to systemic differences
between groups of animals and allowing a more precise
measurement of signalling and muscle hypertrophy in
each individual animal. Additionally, separate animals that
underwent no prior surgery (n = 3 per group) were also
killed to verify that the sham-operated control muscles
truly reflected previously undisturbed resting muscle.
Rats were weighed and anaesthetized with 2–3%
isoflurane and supplemental oxygen for gastrocnemius
ablation. Under aseptic conditions, the distal two-thirds of
the gastrocnemius muscle were surgically removed from
the left hindlimb as previously described (Gordon et al.
2001a,b; Thomson & Gordon, 2005). A sham (control)
operation was performed on the right hindlimb. This
consisted of an incision through the skin and isolation
of the Achilles tendon, but without disruption of the
gastrocnemius muscle. The plantaris muscle from this
limb served as the control. Animals were kept on a
water-circulation heating pad during surgery. Following
each procedure, the incision was closed using stainless steel
surgical clips, and animals were subcutaneously injected
with ∼10 ml of warm saline as well as a one-time
subcutaneous injection of analgesic (Buprenex,
0.03 mg kg−1 body weight). Animals were active
immediately after recovering from anaesthesia, and were
checked twice daily thereafter during the 7 day postoperative period. No signs of postoperative complications
(such as infection or undue distress) were observed.
One week after surgery, the animals were weighed
and anaesthetized with an intraperitoneal injection of
ketamine and xylazine (90 and 10 mg kg−1 body weight,
respectively). The animals were then decapitated, after
which the plantaris muscles from both legs were quickly
removed, trimmed of excess connective tissue, weighed
on an analytical balance, frozen in liquid nitrogen, and
stored at −80◦ C until further analysis.
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Tissue homogenization and determination
of protein concentration
Muscles were homogenized on ice as a 3.5% (w/v)
solution in homogenization buffer (50 mm Hepes,
pH 7.4, 0.1% Triton X-100, 4 mm EGTA, 10 mm EDTA,
15 mm Na4 P2 O7 .10H2 O, 100 mm β-glycerophosphate,
25 mm NaF, 50 μg ml−1 leupeptin, 50 μg ml−1 pepstatin,
33 μg ml−1 aprotinin and 0.5 mm Na3 VO4 ) in a ground
glass homogenizer. An aliquot of homogenate was clarified
by centrifugation for 10 min at 10 000 g, and protein
concentration measurements were made on both the
crude and clarified homogenates using a modified Lowry
procedure (DC Protein Assay; Bio-Rad, Hercules, CA,
USA). Protein assay results from the crude homogenate
were used to calculate total protein per whole muscle (as
previously reported by Thomson & Gordon, 2005), which
allows a more precise estimation of muscle hypertrophy
(protein accretion) than wet weight measurements because
it accounts for potential differences in water weight due
to oedema (Ianuzzo & Chen, 1979). Clarified homogenates were used for Western blotting (preliminary testing
indicated no effect of clarification on Western blotting
results).
SDS-PAGE, Western blotting and immunodetection
Clarified muscle homogenates were solubilized in sample
loading buffer (50 mm Tris-HCl, pH 6.8, 10% glycerol
(v/v), 2% SDS, 2% β-mercaptoethanol, 0.1% bromophenol blue) at a concentration of 1 mg ml−1 and boiled for
5 min. Samples across conditions were equally represented
on each SDS-polyacrylamide gel (7.5% for S6k, eEF2,
mTOR and Akt, 15% for rpS6 and 4E-BP1). Proteins were
separated by electrophoresis and then Western blotted for
1 h at 4◦ C onto a PVDF membrane (Millipore, Bedford,
MA, USA) at 100 V in transfer buffer (25 mm Tris-base,
192 mm glycine, 20% methanol). Visual verification of
transfer and equal protein loading amongst lanes was
accomplished by Ponceau S staining of the membranes.
For immunodetection, membranes were blocked for 1 h
at room temperature in blocking buffer (5% non-fat dry
milk in TBS-T (20 mm Tris-base, 150 mm NaCl, 0.1%
Tween-20), pH 7.6), serially washed in TBS-T at room
temperature, then probed for specific signalling proteins
using antibodies (all from Cell Signalling Technology,
Inc., Beverly, MA, USA) for the detection of Akt,
phospho-Akt (Ser473 ), mTOR, phospho-mTOR (Ser2448 ),
p70S6k, phospho-p70S6k (Thr389 ), phospho-p70S6k
(Thr421 /Ser424 ), rpS6, phospho-rpS6 (Ser235/236 ), 4E-BP1,
eEF2 and phospho-eEF2 (Thr56 ). Regulation at these
residues is considered necessary for full or partial
activation of these proteins (Kimball et al. 1998; Gingras
et al. 1999; Shah et al. 2000; Browne & Proud, 2002).
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D. M. Thomson and S. E. Gordon
Membranes were incubated overnight at 4◦ C in primary
antibody buffer (5.0% BSA in TBS-T, pH 7.6, primary
antibody diluted 1:1000, except for phospho-eEF2 which
was diluted 1:2000), then serially washed in TBS-T,
incubated with horseradish peroxidase (HRP)-conjugated
secondary antibody (Amersham Pharmacia Biotech,
Piscataway, NJ, USA) in blocking buffer for 1 h, and
again serially washed in TBS-T. The HRP activity was
detected using enhanced chemiluminescence reagent
(Femtoglow HRP Substrate Plus; Michigan Diagnostics,
Troy, MI, USA) and exposure to autoradiographic film
(Classic Blue Sensitive; Midwest Scientific, St Louis, MO,
USA). The film was scanned and antigen concentration
was then calculated by quantification of the integrated
optical density (IOD) of the appropriate band(s) using
Gel Pro Analyser software (Media Cybernetics, Silver
Spring, MD, USA). For rpS6 and eEF2, blots were first
probed with the phospho-specific antibody, stripped
with 0.2 m NaOH at room temperature for 5 min, then
reprobed with the antibody that detects total protein
levels. For S6k, blots were probed with the total-S6k
antibody, stripped, probed for phospho-Thr389 S6k,
stripped again, and probed for phospho-Thr421 /Ser424
S6k. Because mTOR protein detection was not effective
after stripping, phospho- and total mTOR values
were obtained from separate blots using the exact
same homogenate-loading buffer preparations and exact
same lane loading. IODs were normalized to the
sham-operated condition of the young adult animals.
Relative phosphorylation for all proteins except 4E-BP1
was determined by normalizing the IOD for the
phospho-protein to the IOD for the corresponding total
protein. Phosphorylation of 4E-BP1 was expressed as
the percentage of the total 4E-BP1 signal that was
encompassed in the slower-migrating, hyperphosphorylated β and γ bands.
To verify that the unilateral ablation procedure
did not alter signalling in the contralateral ‘control’
(sham-operated) muscles, we also compared the signalling
status in the plantaris from the sham-operated leg of the
YA animals with the plantaris muscles from a separate
group of YA animals (n = 3) in which no ablation or sham
surgery had been previously performed. No differences in
protein concentration or phosphorylation were observed
between the above groups for any of the measured
proteins (data not shown). Furthermore, as previously
reported (Thomson & Gordon, 2005), no AMPK protein
concentration or phosphorylation differences existed in
sham-operated plantaris muscles compared to plantaris
muscles from animals experiencing no prior gastrocnemius ablation surgery. These results indicate that the
unilateral ablation procedure did not alter the measured
signalling proteins in contralateral sham-operated
muscles.
J Physiol 574.1
Statistics
Group comparisons were made using analyses of
variance, with repeated measures where appropriate,
and Fisher’s LSD post hoc comparisons when necessary.
Where calculated, average responsiveness for YA and O
animals to the overload stimulus (percentage change
from sham muscle to overloaded muscle within each
individual animal) was compared using Student’s t test. All
correlations were calculated as Pearson product–moment
correlation coefficients. All values are expressed as
means ± s.e.m., and statistical significance was set at a level
of P ≤ 0.05.
Results
Muscle wet weights and total protein contents
All of the muscle wet weight and total protein content
results have been reported previously (Thomson &
Gordon, 2005). Control plantaris muscles from O rats
were significantly atrophied compared to their YA counterparts both in terms of wet weight (321.5 ± 6.7 versus
383.7 ± 12.8 mg) and of protein content (74.0 ± 5.5 versus
109.7 ± 6.5 mg). Overloaded plantaris muscles were also
smaller in O than YA animals (wet weight, 352.9 ±
11.2 versus 497.3 ± 17.7 mg; protein content, 78.4 ± 6.6
versus 130.8 ± 8.7 mg). Although the increase in wet
weight and protein content with overload was significant
in both YA and O muscles, this hypertrophy (expressed
as percentage increase) was significantly diminished in O
compared with YA muscles (wet weight, 9.7 ± 2.3 versus
30.0 ± 4.3%; protein content, 5.6 ± 1.7 versus 19.3 ± 2.9).
Concentration and phosphorylation of Akt
Phosphorylation of Akt at Ser473 is one of two
phosphorylation events involved in the activation of Akt.
Akt phosphorylation status (phospho-Akt/total Akt) at
Ser473 was significantly lower in O control muscles than
in YA control muscles, and decreased with overload in
YA muscles, while no such decrease relative to control
was observed with overload in O muscles (Fig. 1A). The
absolute concentration of phospho-Akt (in contrast to the
Akt phosphorylation status relative to total Akt as reported
above) was elevated with overload in YA and O muscles
without any age-related differences (Fig. 1B). Total Akt
protein concentration was elevated in O compared with
YA muscles, regardless of loading status, and was higher in
overloaded muscles regardless of age (Fig. 1C).
Concentration and phosphorylation of mTOR
While it is not clear what impact mTOR phosphorylation
at Ser2448 has upon its actual activity (Sekulic et al.
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Overload and impaired translational signalling in aged skeletal muscle
J Physiol 574.1
2000), it is generally thought to be stimulatory (Nave
et al. 1999). The phosphorylation status of mTOR
(phospho-mTOR/total mTOR) at Ser2448 was significantly
elevated after overload in YA muscles, while no such
increase relative to control was observed with overload in O muscles (Fig. 2A). Furthermore, although the
absolute concentrations of phospho-mTOR and total
mTOR were elevated after overload in both age groups
(Fig. 2B and C), the average percentage increase in
phospho-mTOR concentration from control to overloaded plantaris muscles was significantly impaired in O
versus YA rats (88 ± 51 versus 292 ± 74%, respectively;
P = 0.042).
Concentration and phosphorylation of S6k
Activation of S6k requires phosphorylation at several
sites. It is generally thought that phosphorylation
at Thr421 /Ser424 (among other sites in a C-terminal
autoinhibitory domain) through poorly understood
Akt Phosphorylation Status
(% of Young Adult Control)
A
mechanisms occurs early in the activation process and
contributes to the ‘opening up’ of the protein and
exposure of other sites, including Thr389 , for subsequent
phosphorylation (Pullen & Thomas, 1997). Of the various
phosphorylation sites, Thr389 is the main site regulated
downstream of mTOR activity (Pearson et al. 1995) and is
most indicative of in vivo S6k activity (Weng et al. 1998).
Phosphorylation status of S6k (phospho-S6k/total S6k)
at Thr389 (Fig. 3A) and Thr421 /Ser424 (Fig. 3B) increased
with overload in YA and O muscles compared with
control muscles. While no age-related differences were
observed for S6k phosphorylation status at Thr421 /Ser424 ,
S6k phosphorylation status at Thr389 was lower in
overloaded O muscles than in overloaded YA muscles.
Differences in absolute concentrations of phospho-S6k
phosphorylated at Thr421 /Ser424 and Thr389 were similar
to those reported above for S6k phosphorylation status,
with O rats demonstrating an impaired phosphorylation
response to overload at Thr389 but not at Thr421 /Ser424
(Fig. 3C and D). Furthermore, absolute concentrations
Young Adult (8 mo.)
Old (30 mo.)
125
100
b
75
a
50
25
0
Control
Overloaded
B
C
P-Akt
S473
300
Total Akt
b
b
200
100
0
Control
Overloaded
Total Akt Protein
(% of Young Adult Control)
Phospho-Akt (Ser473)
(% Young Adult Control)
295
700
a,b
600
b
500
400
a
300
200
100
0
Control
Overloaded
Figure 1. Total and phosphorylated Akt response to overload in young adult and old skeletal muscle
A, plantaris Akt phosphorylation status (phospho-Akt integrated optical density (IOD)/total Akt IOD) at Ser473 is
lower in old (O; 30 months; n = 7) than young adult (YA; 8 months; n = 7) control muscles, and is lower after
1 week of functional overload (unilateral gastrocnemius ablation) in YA, but not in O, rats. B, plantaris phospho-Akt
concentration is elevated after overload regardless of age. C, plantaris total Akt protein concentration is greater
in O compared with YA rats regardless of overload status and is elevated after overload regardless of age. Data
(means ± S.E.M.) are expressed as percentages of YA control (sham-operated) values. a Significant (P ≤ 0.05) effect
of age, b significant effect of overload.
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D. M. Thomson and S. E. Gordon
of phospho-S6k phosphorylated at Thr389 in the overloaded plantaris were significantly correlated with the
degree of hypertrophy (as measured by total muscle
protein content) elicited by overload across all animals
(r = 0.59, P = 0.03; Fig. 7A), and negatively correlated
with the phosphorylation status of AMPK (Thr172 , AMPK
data previously reported Thomson & Gordon, 2005) in the
same muscle (r = − 0.59, P = 0.03; Fig. 7B). Neither the
degree of hypertrophy nor AMPK phosphorylation status,
was correlated with S6k phosphorylated at Thr421 /Ser424
(r = 0.13 and −0.18, respectively). Total S6k concentration
increased in the overloaded muscles of both YA and O rats
compared with control muscles (Fig. 3E).
Concentration and phosphorylation of ribosomal
protein S6
Activation of rpS6 is thought to consist of at least five
phosphorylation events, in a stepwise fashion beginning
mTOR Phosphorylation Status
(% of Young Adult Control)
A
with the phosphorylation of Ser236 and then Ser235 by
S6k (Martin-Perez & Thomas, 1983). Phosphorylation
status of rpS6 (phospho-rpS6/total rpS6) at Ser235/236
was elevated with overload, regardless of age, and was
significantly lower in O muscles than in YA muscles
independent of overload status (Fig. 4A). Both the absolute
concentration of phospho-rpS6 and total rpS6 protein
concentration were elevated with overload independent
of age (Fig. 4B and C).
Concentration and phosphorylation of eEF2
Phosphorylation of eEF2 at Thr56 is inhibitory on
its activity (Browne & Proud, 2002). No significant
effects of age or overload were observed on eEF2
phosphorylation status (phospho-eEF2/total eEF2) at
Thr56 (Fig. 5A), although there was a non-significant trend
for eEF2 phosphorylation status to increase with overload in O muscles (P = 0.13). Absolute concentrations
Young Adult (8 mo.)
Old (30 mo.)
250
b
200
150
a
100
50
0
Control
Overloaded
B
C
P-mTOR
S2448
Total mTOR
b
400
b
300
200
a
100
0
Control
Overloaded
Total mTOR Protein
(% of Young Adult Control)
Phospho-mTOR (S2448)
(% of Young Adult Control)
J Physiol 574.1
b
350
300
b
250
200
150
100
50
0
Control
Overloaded
Figure 2. Total and phosphorylated mTOR response to overload in young adult and old skeletal muscle
A, plantaris mammalian target of rapamycin (mTOR) phosphorylation status (phospho-mTOR IOD/total mTOR IOD)
at Ser2448 is elevated after 1 week of functional overload compared to control (sham-operated) conditions in YA
(n = 7), but not O (n = 7) muscles. B, plantaris phospho-mTOR concentration is elevated after overload regardless of
age (but note that the average percentage increase in phospho-mTOR concentration from control to overloaded
plantaris muscles was significantly less O versus YA rats, 88 ± 51 versus 292 ± 74%, respectively; P = 0.042).
C, plantaris mTOR protein concentration is elevated after overload regardless of age. Data (means ± S.E.M.) are
expressed as percentages of YA control (sham-operated) values. b Significant (P ≤ 0.05) effect of overload.
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J Physiol 574.1
Overload and impaired translational signalling in aged skeletal muscle
of phospho-eEF2 increased in the overloaded muscles
in both age groups, and were also higher in O versus
YA in both control and overloaded muscles (Fig. 5B).
Interestingly, total eEF2 concentration increased with
overload in YA, but not O muscles (Fig. 5C). The average
intra-animal increase of total eEF2 concentration in the
overloaded plantaris relative to that of the control leg
was also significantly greater for YA rats than for O rats
(38.9 ± 9.8 versus 5.6 ± 5.5%, respectively; P = 0.01).
Additionally, this percentage increase in total eEF2 protein
concentration was strongly correlated across ages with the
percent hypertrophy observed in the plantaris (r = 0.84,
P = 0.0002; Fig. 7C), and negatively correlated with
the phosphorylation status (Thr172 ) of AMPK in these
Figure 3. Total and phosphorylated S6k response to overload in young adult and old skeletal muscle
A, plantaris 70 kDa ribosomal protein S6 kinase (S6k) phosphorylation status (phospho-S6k IOD/total S6k IOD) at
Thr389 is elevated after 1 week of functional overload compared with control (sham-operated) conditions in YA
(n = 7) and O (n = 7) muscles, but is lower after overload in O than in YA muscles. B, plantaris S6k phosphorylation
status at Thr421 /Ser424 is elevated after overload compared with control (sham-operated) conditions regardless of
age. C, plantaris phospho-S6k (Thr389 ) concentration is elevated after overload in YA and O muscles, but is
lower after overload in O than in YA muscles. D, plantaris phospho-S6k (Thr421 /Ser424 ) concentration is elevated
after overload regardless of age. E, plantaris total S6k protein is elevated after overload regardless of age. Data
(means ± S.E.M.) are expressed as percentages of YA control (sham-operated) values. a Significant (P ≤ 0.05) effect
of age, b significant effect of overload.
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overloaded plantaris muscles (r = −0.63, P = 0.02;
Fig. 7D) (AMPK data previously reported by Thomson
& Gordon, 2005).
Concentration and phosphorylation of 4E-BP1
4E-BP1 phosphorylation status (phospho-4E-BP1/total
4E-BP1) was increased in the overloaded muscles of both
YA and O rats, but was significantly lower with overload in the O than in the YA muscles (Fig. 6A). Total
4E-BP1 protein concentration was increased with overload but was not different between YA and O muscles
(Fig. 6B). Phosphorylation of 4E-BP1 occurs on at least
seven sites (Wang et al. 2005) which leads to its
dissociation from the translation initiation factor eIF4E
and therefore promotes enhanced cap-dependent protein
translation. Though additional kinases are necessary
for full phosphorylation and activation of 4E-BP1,
phosphorylation of the protein by mTOR is thought
to be crucial in the initial events that ‘prime’ 4E-BP1
for phosphorylation by other proteins (Gingras et al.
1999), and may regulate later phosphorylation events
rpS6 Phosphorylation Status
(% of Young Adult Control)
A
as well (Wang et al. 2005). In this investigation, the
phosphorylation status of 4E-BP1 was calculated as the
percentage of the total 4E-BP1 that was encompassed in
the slower-migrating, hyperphosphorylated β and γ
bands (Fig. 6B). Although the true relationship between
each specific hyperphosphorylated 4E-BP1 band and
resultant 4E-BP1 activity has not been definitively
established to our knowledge (e.g. it is not known which
bands are representative of increased eIF4E activity), it
is generally believed that 4E-BP1 hyperphosphorylation
results in increased dissociation from eIF4E and thus
increased eIF4E activation and translation initiation
(Gingras et al. 1999; Shah et al. 2000). Accordingly, we
observed that the phosphorylation of 4E-BP1 (percentage
of 4E-BP1 signal encompassed in the β and γ bands) in
the overloaded plantaris muscles was positively correlated
with percentage plantaris hypertrophy across all animals
(r = 0.66, P = 0.01; Fig. 7E), and negatively associated
with the phosphorylation status of AMPK (Thr172 , AMPK
data previously reported Thomson & Gordon, 2005) in the
same muscle (strong but non-significant trend: r = −0.53,
P = 0.051; Fig. 7F).
Young Adult (8 mo.)
Old (30 mo.)
300
b
a,b
200
a
100
0
Control
Overloaded
B
C
P-rpS6
S235/S236
Total rpS6
b
900
800
700
600
500
400
300
200
100
0
b
a,b
Control
Overloaded
Total rpS6 Protein
(% of Young Adult Control)
Phospho-rpS6 (S235/S236)
(% of Young Adult Control)
J Physiol 574.1
b
350
b
300
250
200
150
100
50
0
Control
Overloaded
Figure 4. Total and phosphorylated rpS6 response to overload in young adult and old skeletal muscle
A, plantaris ribosomal protein S6 (rpS6) phosphorylation status (phospho-rpS6 IOD/total rpS6 IOD) at Ser235/236 is
lower in O (n = 7) than YA (n = 7) rats and is elevated after 1 week of functional overload regardless of age.
B, plantaris phospho-rpS6 concentration is elevated after overload regardless of age. C, plantaris total rpS6
concentration is elevated after overload regardless of age. Data (means ± S.E.M.) are expressed as percentages
of YA control (sham-operated) values. a Significant (P ≤ 0.05) effect of age, b significant effect of overload.
C 2006 The Authors. Journal compilation C 2006 The Physiological Society
Overload and impaired translational signalling in aged skeletal muscle
J Physiol 574.1
Discussion
We and others have previously shown that fast-twitch
muscle protein synthesis (Pehme et al. 2004b) and growth
(Blough & Linderman, 2000; Alway et al. 2002; Degens
& Alway, 2003; Pehme et al. 2004b; Thomson & Gordon,
2005) are reduced in old rats compared with young rats
after ablation, tenotomy or denervation of a synergistic
muscle. It is doubtful that these results solely reflect a
delayed response with age, as some have observed highly
diminished growth in aged muscle (despite robust growth
in young muscle) by using the same model as that used
in the current investigation over a much longer overload
period (8 weeks versus 1 week) (Blough & Linderman,
2000). Because of their importance in the hypertrophic
response of skeletal muscle to chronic overload (Baar
& Esser, 1999; Bodine et al. 2001), we compared the
contents and phosphorylation states of mTOR and several
of its downstream targets in YA and O rat fast-twitch
plantaris muscle after 1 week of an overload-induced
growth stimulus. Ours is the first report of deficits in
the phosphorylation states of mTOR, S6k, rpS6 and
Young Adult (8 mo.)
Old (30 mo.)
250
200
150
100
a
50
0
B
Phospho-eEF2 (T56)
(% of Young Adult Control)
4E-BP1, as well as deficits in the accretion of eEF2 total
protein, in conjunction with impaired overload-induced
growth in aged fast-twitch skeletal muscle. Moreover,
when compared with previously reported AMPK data
from the same animals (Thomson & Gordon, 2005), our
results also indicate that this age-related suppression of
fast-twitch muscle translational signalling under chronic
overload conditions may be at least partly related to AMPK
hyperactivation.
Our current results confirm those of others (Bodine
et al. 2001; Reynolds et al. 2002), who demonstrated
the importance of mTOR-S6k signalling in the hypertrophic response of chronically overloaded young muscle.
However, we now extend upon those results to show
site-specific increases in S6k phosphorylation at Thr389 and
Thr421 /Ser424 as well as an increase in rpS6 phosphorylation
at Ser235/236 with overload. We also show that S6k
phosphorylation at Thr389 (an mTOR-specific residue;
Pearson et al. 1995; Burnett et al. 1998) was significantly
correlated with degree of muscle hypertrophy in overloaded muscles, while phosphorylation at Thr421 /Ser424
was not. Our data further elucidate findings that increased
Control
Overloaded
C
P-eEF2
T56
Tot eEF2
Total
250
a,b
200
a
b
150
a,b
100
50
0
Control
Overloaded
Total eEF2 Protein
(% of Young Adult Control)
eEF2 Phosphorylation Status
(% of Young Adult Control)
A
299
200
b
150
100
50
0
Control
Overloaded
Figure 5. Total and phosphorylated eEF2 response to overload in young adult and old skeletal muscle
A, plantaris eukaryotic elongation factor 2 (eEF2) phosphorylation status (phospho-eEF2 IOD/total eEF2 IOD) at
Thr56 is not statistically different between O (n = 7) and YA (n = 7) rats or changed with 1 week of functional
overload. B, plantaris phospho-eEF2 concentration is greater in O compared with YA rats regardless of overload
status and is elevated after overload regardless of age. C, plantaris eEF2 protein concentration is elevated after
overload in YA, but not O rats. Data (means ± S.E.M.) are expressed as percentages of YA control (sham-operated)
values. a Significant (P ≤ 0.05) effect of age, b significant effect of overload.
C 2006 The Authors. Journal compilation C 2006 The Physiological Society
300
D. M. Thomson and S. E. Gordon
S6k phosphorylation at unspecified residues (multiple
bands assessed by gel shifts) is significantly correlated
with muscle hypertrophy elicited by chronic electrical
stimulation (Baar & Esser, 1999). The fact that the
mTOR (Parkington et al. 2003), S6k (Baar & Esser,
1999), and hypertrophy (Baar & Esser, 1999) responses to
electrical stimulation are all greater in fast-twitch (versus
slow-twitch) muscle further confirms the importance of
mTOR-S6k signalling in mechanically induced skeletal
muscle growth. Not surprisingly, mTOR and/or S6k
phosphorylations are decreased during unloading- or
immobilization-induced atrophy in young adult (Bodine
et al. 2001; Reynolds et al. 2002) and old (Morris et al.
2004) muscle, although others have reported no effect of
immobilization on S6k phosphorylation in young adult
muscle (Childs et al. 2003).
4E-BP1 Phosphorylation Status
(% in β + γ isoform)
A
Young Adult (8 mo.)
Old (30 mo.)
100
75
b
a,b
50
25
0
Control
Overloaded
B
γ
β
α
Total 4E-BP1
Total 4E-BP1 Protein
(% of Young Adult Control)
350
b
b
300
250
200
150
100
50
0
Control
Overloaded
Figure 6. Total and phosphorylated 4E-BP1 response to
overload in young adult and old skeletal muscle
A, plantaris eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)
phosphorylation status (expressed as a percentage of total 4E-BP1
signal encompassed in the slower-migrating, hyperphosphorylated β
and γ bands) is elevated after 1 week of functional overload
compared with control (sham-operated) conditions in YA (n = 7) and
O (n = 7) muscles, but is lower after overload in O than in YA muscles.
B, plantaris 4E-BP1 total protein concentration is elevated after
overload regardless of age. Data (means ± S.E.M.) are expressed as
percentages of YA, control (sham-operated) values. a Significant
(P ≤ 0.05) effect of age, b significant effect of overload.
J Physiol 574.1
In stark contrast to the anabolic response of mTOR,
S6k and rpS6 to overload in young adult muscle,
the phosphorylation states of mTOR, S6k (at Thr389
only), and rpS6 were significantly lower in the
overloaded muscles of O compared with YA rats
(although S6k and rpS6 phosphorylation states were
still elevated above O control muscle). The attenuated
phosphorylation responses of mTOR and S6k (Thr389 )
to chronic overload in the present investigation agree
with the finding that S6k phosphorylation at Thr389 is
attenuated in old slow-twitch muscle when stimulated
with reloading after immobilization (Morris et al. 2004),
as well as reports that mTOR/S6k responsiveness may
potentially be reduced after acute contractions in old
fast-twitch muscle (Parkington et al. 2003, 2004). The
present investigation further advances the developing
paradigm that mTOR dysregulation (as opposed to other
signalling pathways) may play a central role in impaired
translational signalling in ageing skeletal muscle, because
the diminished responsiveness to muscle overload of S6k
phosphorylation at Thr389 (but not at Thr421 /Ser424 ) agrees
with the fact that mTOR phosphorylation in response to
overload was also diminished with age. Of the various
sites, Thr389 is most indicative of in vivo S6k activity (Weng
et al. 1998; Isotani et al. 1999) and is the primary site
phosphorylated by mTOR (Pearson et al. 1995; Burnett
et al. 1998). This is further supported by our finding that
S6k phosphorylation at Thr389 (but not at Thr421 /Ser424 )
was correlated to the hypertrophic response in
overloaded muscles. Finally, the potential for diminished
rpS6 signalling with age in overloaded skeletal muscle
has not been previously examined to our knowledge.
Interestingly, the fact that rpS6 phosphorylation
at Ser235/236 was reduced with age in both overloaded and control muscles (while upstream S6k Thr389
phosphorylation was only reduced in overloaded muscles)
indicates that resting muscle rpS6 phosphorylation at
Ser235/236 may also be regulated by mechanisms other
than S6k, similar to other tissue types (Martin-Perez
& Thomas, 1983; Pende et al. 2004). Nevertheless, our
results collectively indicate that impaired S6k activation by
mTOR (but perhaps not S6k activation by other pathways)
may be a major factor underlying the diminished protein
synthesis and growth with age in overloaded fast-twitch
skeletal muscle (Pehme et al. 2004b).
In the young adult animals, the overload-induced
increase in total eEF2 protein concentration in the
absence of a concomitant increase in eEF2 relative
phosphorylation status (per total eEF2 protein) at Thr56
(which is inhibitory upon eEF2 activity; Browne & Proud,
2002) appears to indicate an increased drive for protein
elongation because of the presumed resultant increase
in unphosphorylated (active) eEF2. On the contrary,
old muscles demonstrated no such anabolic increase in
total eEF2 content. Further, old animals demonstrated a
C 2006 The Authors. Journal compilation C 2006 The Physiological Society
J Physiol 574.1
Overload and impaired translational signalling in aged skeletal muscle
higher phospho-eEF2 content versus young adult animals
regardless of overload status as well as a slight (albeit
non-significant; P = 0.13) overload-induced increase in
eEF2 phosphorylation status (relative to total eEF2).
The higher eEF2 phosphorylation in aged fast-twitch
muscle is interesting, because S6k normally acts to inhibit
eEF2 phosphorylation (and thus increase its activity)
through inhibition of eEF2 kinase (eEF2k) (Wang et al.
2001; Browne & Proud, 2004). Others have recently
reported an acute increase in eEF2 phosphorylation at
Figure 7. Relationships of selected variables to muscle hypertrophy and AMPK phosphorylation status
A and B, phospho-S6k (Thr389 ) content is positively correlated with percentage hypertrophy, and negatively
correlated with 5 -AMP-activated protein kinase (AMPK) phosphorylation status (Thr172 ) in overloaded fast-twitch
plantaris muscles of YA (•) and O () rats (n = 7 per group). Note that no such correlations existed between
phospho-S6k at Thr421 /Ser424 and either AMPK phosphorylation status or percentage hypertrophy. C and
D, accretion of eEF2 protein content is positively correlated with percentage hypertrophy, and negatively correlated
with AMPK phosphorylation status in overloaded fast-twitch plantaris muscles of young adult and old rats.
E and F, phosphorylation of 4E-BP1 (percentage of 4E-BP1 signal encompassed in the β and γ bands) is positively
correlated with percentage hypertrophy, and negatively associated (P = 0.051) with AMPK phosphorylation status
in overloaded fast-twitch plantaris muscles of YA and O rats.
C 2006 The Authors. Journal compilation C 2006 The Physiological Society
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302
D. M. Thomson and S. E. Gordon
Thr56 in response to aerobic exercise (Rose et al. 2005),
which does not elicit hypertrophy; however, the current
investigation is the first to examine the response of eEF2 to
hypertrophic skeletal muscle overload. The increased total
eEF2 content in young but not aged fast-twitch muscles
in the current investigation suggests that total eEF2 may
play an important role in the anabolic response of skeletal
muscle to overload. Accordingly, we found a strong
significant correlation between the increase in eEF2 total
protein concentration and the degree of hypertrophy in
the overloaded muscles. Notably, some (Terada et al. 1994;
Jefferies et al. 1997), but not all (Stolovich et al. 2002;
Pende et al. 2004), studies have also linked S6k and rpS6
to the selective translation of mRNAs with a 5 -terminal
polypyrimidine (5 TOP) tract, which encode ribosomal
proteins and elongation factors including eEF2. Thus,
the loss of an overload-induced increase in eEF2 total
protein concentration in old muscles could be attributable
to their diminished mTOR/S6k/rpS6 signalling. More
research is warranted to examine the possibility that
eEF2 content is upregulated by mTOR/S6k/rpS6 signalling
in overloaded young fast-twitch muscle, as well as
the intriguing possibility that this response is lost
in old muscle due to diminished mTOR/S6k/rpS6
signalling.
Similar to mTOR and S6k (Thr389 ) phosphorylation,
the 4E-BP1 phosphorylation response to overload, though
still significant, was greatly reduced in our aged muscles
compared to those of young adult animals. These
findings further suggest that mTOR signalling is deficient
in old fast-twitch muscles under overload conditions,
as both S6k (Thr389 ) and 4E-BP1 lie independently
downstream of mTOR and are considered reliable
indicators of in vivo mTOR activity (Hay & Sonenberg,
2004). Because it binds to eIF4E and prevents it
from forming the eIF4F initiation complex with eIF4A
and eIF4G, unphosphorylated 4E-BP1 is an important
regulatory protein inhibiting translation initiation (Nader
et al. 2002; Bolster et al. 2003a). The fact that overload
elicited an increased 4E-BP1 phosphorylation status in
our young adult muscles (resulting from a greater increase
in phospho-4E-BP1 relative to total 4E-BP1) indicates a
potential freeing of eIF4E to participate in eIF4F formation
and initiation of protein translation in 7 day overloaded
plantaris muscles of young adult rats. These results
agree with observations of decreased 4E–BP1–eIF4E
association and increased eIF4E–eIF4G association in
overloaded plantaris muscles of young adult rats 14 days
after synergist ablation, effects that were both blocked by
rapamycin inhibition of mTOR (Bodine et al. 2001). Thus,
increased 4E-BP1 phosphorylation in overloaded young
adult fast-twitch muscle appears to be mTOR-dependent,
which may explain the reduced 4E-BP1 phosphorylation
(concomitant with diminished mTOR phosphorylation)
in response to overload in our old muscles. Our current
J Physiol 574.1
data also further show that 4E-BP1 phosphorylation
in overloaded muscles is significantly correlated with
hypertrophy in those muscles, and implicate the impaired
4E-BP1 phosphorylation response to muscle overload in old animals as another potential mechanism
underlying the diminished growth observed in aged
fast-twitch muscle.
In our overloaded muscles, the age-related impairments
in the phosphorylation states of mTOR and downstream
translational signalling intermediates (S6k, rpS6 and
4E-BP1), along with deficits in eEF2 protein accretion,
indicate that mTOR dysregulation may play a primary role
on several levels in the diminished protein synthesis and
growth response to mechanical loading in aged fast-twitch
muscle (Tamaki et al. 2000; Pehme et al. 2004b; Thomson
& Gordon, 2005). However, the upstream mediator(s) in
aged fast-twitch muscle that may lead to impaired mTOR
signalling under such conditions remain to be identified.
To this end, we chose to examine the potential relationship
of this diminished translational signalling to AMPK, which
is known to inhibit S6k, 4E-BP1 and eEF2 signalling and
protein synthesis in resting young skeletal and/or cardiac
muscle (Bolster et al. 2002; Horman et al. 2002; Chan et al.
2004), probably via its inhibition of mTOR (Cheng et al.
2004) as well as its mTOR-independent inhibition of eEF2
via eEF2 kinase (Browne et al. 2004). AMPK is activated by
cellular energy depletion, which is exacerbated in old versus
young skeletal muscle at rest and with exercise (Bastien
& Sanchez, 1984; Marcinek et al. 2005). Accordingly, we
recently reported (using the same animals and muscles
as in the current study) that AMPK phosphorylation
(Thr172 ) is significantly elevated with age in resting and
overloaded plantaris muscles (Thomson & Gordon, 2005).
Moreover, there was a strong negative correlation between
the degree of AMPK phosphorylation and the degree
of hypertrophy in the overloaded muscles, implicating
AMPK as a potentially important negative regulator of
overload-induced skeletal muscle hypertrophy (Thomson
& Gordon, 2005).
Correlational results in the current investigation raise
the possibility that AMPK hyperphosphorylation may be
a potential upstream mechanism by which translational
signalling and overload-induced growth is suppressed
in aged fast-twitch muscles. Using AMPK data previously obtained from the same muscles (Thomson &
Gordon, 2005), we found that AMPK phosphorylation
state was negatively correlated with the amount of S6k
phosphorylated at the mTOR-specific Thr389 residue,
but not at Thr421 /Ser424 . This fits with previously
reported data showing that AMPK activation attenuates
amino-acid-stimulated phosphorylation of S6k at Thr389 ,
but not at Thr421 /Ser424 (Krause et al. 2002; Moller et al.
2004). Similar to S6k, a negative relationship (P = 0.051)
was also observed between the phosphorylation statuses of
AMPK and 4E-BP1. Because S6k (Thr389 ) and 4E-BP1 are
C 2006 The Authors. Journal compilation C 2006 The Physiological Society
J Physiol 574.1
Overload and impaired translational signalling in aged skeletal muscle
both independent targets of mTOR (Hay & Sonenberg,
2004), it is possible that AMPK inhibited S6k (Thr389 )
and 4E-BP1 phosphorylation in these muscles via its
reported suppression of mTOR phosphorylation (Bolster
et al. 2002). Additionally, the negative correlation between
AMPK phosphorylation status and the percentage increase
in eEF2 content in the overloaded muscles points to the
possibility that AMPK may also somehow suppress total
eEF2 protein accretion, possibly by suppressing S6k and
its specific targeting of 5 TOP mRNA translation (Terada
et al. 1994; Jefferies et al. 1997), which includes eEF2.
Overall, such correlative evidence is intriguing when taken
in concert with the fact that AMPK is hyperphosphorylated
in aged fast-twitch muscle (Thomson & Gordon, 2005) and
is also known to negatively regulate translational signalling
and protein synthesis in resting skeletal muscle (Bolster
et al. 2002). Nevertheless, more research is warranted to
establish any potential causal relationship between AMPK
hyperactivation and suppressed translational signalling
and growth with overload in aged fast-twitch muscles.
In conclusion, here we have reported evidence of
impaired phosphorylation states of mTOR and its
downstream translational signalling intermediates S6k
(specifically at the mTOR-specific Thr389 residue but
not at Thr421 /Ser424 ), rpS6, and 4E-BP1 in overloaded
fast-twitch skeletal muscles of old rats, as well as
deficits in the accretion of eEF2 total protein in the
same muscles (further indicating impaired S6k and rpS6
signalling). These findings are novel, as they are the first
to demonstrate impaired translational signalling in direct
conjunction with diminished overload-induced growth in
aged skeletal muscle. Moreover, our data also raise the
interesting possibility that AMPK hyperactivation may
lie upstream of this suppressed translational signalling
with age in overloaded fast-twitch muscle, perhaps acting
via an mTOR-dependent mechanism. Further work is
necessary to establish the causality of AMPK in this
respect. Regardless of the upstream mechanism(s), the
results of this investigation provide solid evidence that
the impaired overload-induced growth observed in old
fast-twitch muscle may be closely related to underlying
decrements in signalling pathways controlling protein
translation.
References
Alway SE, Degens H, Krishnamurthy G & Smith CA (2002).
Potential role for Id myogenic repressors in apoptosis and
attenuation of hypertrophy in muscles of aged rats.
Am J Physiol Cell Physiol 283, C66–C76.
Armstrong RB & Phelps RO (1984). Muscle fiber type
composition of the rat hindlimb. Am J Anat 171,
259–272.
Baar K & Esser K (1999). Phosphorylation of p70 (S6k)
correlates with increased skeletal muscle mass following
resistance exercise. Am J Physiol 276, C120–C127.
C 2006 The Authors. Journal compilation C 2006 The Physiological Society
303
Baillie AG & Garlick PJ (1991). Protein synthesis in adult
skeletal muscle after tenotomy: responses to fasting and
insulin infusion. J Appl Physiol 71, 1020–1024.
Bastien C & Sanchez J (1984). Phosphagens and glycogen
content in skeletal muscle after treadmill training in young
and old rats. Eur J Appl Physiol 52, 291–295.
Blough ER & Linderman JK (2000). Lack of skeletal muscle
hypertrophy in very aged male Fischer 344 × Brown Norway
rats. J Appl Physiol 88, 1265–1270.
Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL,
Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC,
Glass DJ & Yancopoulos GD (2001). Akt/mTOR pathway is a
crucial regulator of skeletal muscle hypertrophy and can
prevent muscle atrophy in vivo. Nat Cell Biol 3, 1014–1019.
Bolster DR, Crozier SJ, Kimball SR & Jefferson LS (2002).
AMP-activated protein kinase suppresses protein synthesis in
rat skeletal muscle through down-regulated mammalian
target of rapamycin (mTOR) signaling. J Biol Chem 277,
23977–23980.
Bolster DR, Kimball SR & Jefferson LS (2003a). Translational
control mechanisms modulate skeletal muscle gene
expression during hypertrophy. Exerc Sport Sci Rev 31,
111–116.
Bolster DR, Kubica N, Crozier SJ, Williamson DL, Farrell PA,
Kimball SR & Jefferson LS (2003b). Immediate response of
mammalian target of rapamycin (mTOR)-mediated
signalling following acute resistance exercise in rat skeletal
muscle. J Physiol 553, 213–220.
Browne GJ, Finn SG & Proud CG (2004). Stimulation of the
AMP-activated protein kinase leads to activation of
eukaryotic elongation factor 2 kinase and to its
phosphorylation at a novel site, serine 398. J Biol Chem 279,
12220–12231.
Browne GJ & Proud CG (2002). Regulation of peptide-chain
elongation in mammalian cells. Eur J Biochem 269,
5360–5368.
Browne GJ & Proud CG (2004). A novel mTOR-regulated
phosphorylation site in elongation factor 2 kinase modulates
the activity of the kinase and its binding to calmodulin. Mol
Cell Biol 24, 2986–2997.
Burnett PE, Barrow RK, Cohen NA, Snyder SH & Sabatini DM
(1998). RAFT1 phosphorylation of the translational
regulators p70, S6 kinase and 4E-BP1. Proc Natl Acad Sci U S
A 95, 1432–1437.
Chan AY, Soltys CL, Young ME, Proud CG & Dyck JR (2004).
Activation of AMP-activated protein kinase inhibits protein
synthesis associated with hypertrophy in the cardiac
myocyte. J Biol Chem 279, 32771–32779.
Cheng SW, Fryer LG, Carling D & Shepherd PR (2004).
Thr2446 is a novel mammalian target of rapamycin (mTOR)
phosphorylation site regulated by nutrient status. J Biol
Chem 279, 15719–15722.
Childs TE, Spangenburg EE, Vyas DR & Booth FW (2003).
Temporal alterations in protein signaling cascades during
recovery from muscle atrophy. Am J Physiol Cell Physiol 285,
C391–C398.
Cuthbertson D, Smith K, Babraj J, Leese G, Waddell T,
Atherton P, Wackerhage H, Taylor PM & Rennie MJ (2005).
Anabolic signaling deficits underlie amino acid resistance of
wasting, aging muscle. FASEB J 19, 422–424.
304
D. M. Thomson and S. E. Gordon
Dardevet D, Sornet C, Balage M & Grizard J (2000).
Stimulation of in vitro rat muscle protein synthesis by
leucine decreases with age. J Nutr 130, 2630–2635.
Degens H & Alway SE (2003). Skeletal muscle function and
hypertrophy are diminished in old age. Muscle Nerve 27,
339–347.
Gingras AC, Gygi SP, Raught B, Polakiewicz RD, Abraham RT,
Hoekstra MF, Aebersold R & Sonenberg N (1999).
Regulation of 4E-BP1 phosphorylation: a novel two-step
mechanism. Genes Dev 13, 1422–1437.
Gordon SE, Davis BS, Carlson CJ & Booth FW (2001a). ANG II
is required for optimal overload-induced skeletal muscle
hypertrophy. Am J Physiol Endocrinol Metab 280, E150–E159.
Gordon SE, Fluck M & Booth FW (2001b). Selected
contribution: Skeletal muscle focal adhesion kinase, paxillin,
and serum response factor are loading dependent. J Appl
Physiol 90, 1174–1183, discussion 1165.
Guillet C, Prod’homme M, Balage M, Gachon P, Giraudet C,
Morin L, Grizard J & Boirie Y (2004). Impaired anabolic
response of muscle protein synthesis is associated with
S6K1 dysregulation in elderly humans. FASEB J 18,
1586–1587.
Häkkinen K, Kraemer WJ, Newton RU & Alen M (2001).
Changes in electromyographic activity, muscle fibre and
force production characteristics during heavy resistance/
power strength training in middle-aged and older men and
women. Acta Physiol Scand 171, 51–62.
Häkkinen K, Newton RU, Gordon SE, McCormick M, Volek JS,
Nindl BC, Gotshalk LA, Campbell WW, Evans WJ,
Häkkinen A, Humphries BJ & Kraemer WJ (1998). Changes
in muscle morphology, electromyographic activity, and force
production characteristics during progressive strength
training in young and older men. J Gerontol A Biol Sci Med
Sci 53, B415–B423.
Hay N & Sonenberg N (2004). Upstream and downstream of
mTOR. Genes Dev 18, 1926–1945.
Hernandez JM, Fedele MJ & Farrell PA (2000). Time course
evaluation of protein synthesis and glucose uptake after
acute resistance exercise in rats. J Appl Physiol 88,
1142–1149.
Horman S, Browne G, Krause U, Patel J, Vertommen D,
Bertrand L, Lavoinne A, Hue L, Proud C & Rider M (2002).
Activation of AMP-activated protein kinase leads to the
phosphorylation of elongation factor 2 and an inhibition of
protein synthesis. Curr Biol 12, 1419–1423.
Ianuzzo CD & Chen V (1979). Metabolic character of
hypertrophied rat muscle. J Appl Physiol 46, 738–742.
Isotani S, Hara K, Tokunaga C, Inoue H, Avruch J & Yonezawa
K (1999). Immunopurified mammalian target of rapamycin
phosphorylates and activates p 70 S6 kinase alpha in vitro.
J Biol Chem 274, 34493–34498.
Jefferies HB, Fumagalli S, Dennis PB, Reinhard C, Pearson RB
& Thomas G (1997). Rapamycin suppresses 5 TOP mRNA
translation through inhibition of p70s6k. EMBO J 16,
3693–3704.
Kimball SR, Horetsky RL & Jefferson LS (1998). Signal
transduction pathways involved in the regulation of protein
synthesis by insulin in L6 myoblasts. Am J Physiol 274,
C221–C228.
J Physiol 574.1
Kimball SR, O’Malley JP, Anthony JC, Crozier SJ & Jefferson LS
(2004). Assessment of biomarkers of protein anabolism in
skeletal muscle during the life span of the rat: sarcopenia
despite elevated protein synthesis. Am J Physiol Endocrinol
Metab 287, E772–E780.
Krause U, Bertrand L & Hue L (2002). Control of p70
ribosomal protein S6 kinase and acetyl-CoA carboxylase by
AMP-activated protein kinase and protein phosphatases in
isolated hepatocytes. Eur J Biochem 269, 3751–3759.
Li M, Li C & Parkhouse WS (2003). Age-related differences in
the des IGF-I-mediated activation of Akt-1 and p70 S6K in
mouse skeletal muscle. Mech Ageing Dev 124, 771–778.
Marcinek DJ, Schenkman KA, Ciesielski WA, Lee D & Conley
KE (2005). Reduced mitochondrial coupling in vivo alters
cellular energetics in aged mouse skeletal muscle. J Physiol
569, 467–473.
Martin-Perez J & Thomas G (1983). Ordered phosphorylation
of 40S ribosomal protein S6 after serum stimulation of
quiescent 3T3 cells. Proc Natl Acad Sci U S A 80, 926–930.
Moller MT, Samari HR & Seglen PO (2004). Toxin-induced tail
phosphorylation of hepatocellular S6 kinase: evidence for a
dual involvement of the AMP-activated protein kinase in S6
kinase regulation. Toxicol Sci 82, 628–637.
Morris RT, Spangenburg EE & Booth FW (2004).
Responsiveness of cell signaling pathways during the failed
15-day regrowth of aged skeletal muscle. J Appl Physiol 96,
398–404.
Nader GA & Esser KA (2001). Intracellular signaling specificity
in skeletal muscle in response to different modes of exercise.
J Appl Physiol 90, 1936–1942.
Nader GA, Hornberger TA & Esser KA (2002). Translational
control: implications for skeletal muscle hypertrophy. Clin
Orthop Relat Res 403 Suppl, S178–187.
Nave BT, Ouwens M, Withers DJ, Alessi DR & Shepherd PR
(1999). Mammalian target of rapamycin is a direct target for
protein kinase B: identification of a convergence point for
opposing effects of insulin and amino-acid deficiency on
protein translation. Biochem J 344, 427–431.
Parkington JD, LeBrasseur NK, Siebert AP & Fielding RA
(2004). Contraction-mediated mTOR, p70S6k, and ERK1/2
phosphorylation in aged skeletal muscle. J Appl Physiol 97,
243–248.
Parkington JD, Siebert AP, LeBrasseur NK & Fielding RA
(2003). Differential activation of mTOR signaling by
contractile activity in skeletal muscle. Am J Physiol Regul
Integr Comp Physiol 285, R1086–R1090.
Pearson RB, Dennis PB, Han JW, Williamson NA, Kozma SC,
Wettenhall RE & Thomas G (1995). The principal target of
rapamycin-induced p70s6k inactivation is a novel
phosphorylation site within a conserved hydrophobic
domain. EMBO J 14, 5279–5287.
Pehme A, Alev K, Kaasik P, Julkunen A & Seene T (2004a). The
effect of mechanical loading on the MyHC synthesis rate and
composition in rat plantaris muscle. Int J Sports Med 25,
332–338.
Pehme A, Alev K, Kaasik P & Seene T (2004b). Age-related
changes in skeletal-muscle myosin heavy-chain composition:
effect of mechanical loading. J Aging Phys Act 12,
29–44.
C 2006 The Authors. Journal compilation C 2006 The Physiological Society
J Physiol 574.1
Overload and impaired translational signalling in aged skeletal muscle
Pende M, Um SH, Mieulet V, Sticker M, Goss VL, Mestan J,
Mueller M, Fumagalli S, Kozma SC & Thomas G (2004).
S6K1 (−/−)/S6K2 (−/−) mice exhibit perinatal lethality and
rapamycin-sensitive 5 -terminal oligopyrimidine mRNA
translation and reveal a mitogen-activated protein
kinase-dependent S6 kinase pathway. Mol Cell Biol 24,
3112–3124.
Pullen N & Thomas G (1997). The modular phosphorylation
and activation of p70s6k. FEBS Lett 410, 78–82.
Reynolds THT, Bodine SC & Lawrence JC Jr (2002). Control of
Ser2448 phosphorylation in the mammalian target of
rapamycin by insulin and skeletal muscle load. J Biol Chem
277, 17657–17662.
Rose AJ, Broholm C, Kiillerich K, Finn SG, Proud CG, Rider
MH, Richter EA & Kiens B (2005). Exercise rapidly increases
eukaryotic elongation factor 2 phosphorylation in skeletal
muscle of men. J Physiol 569, 223–228.
Sekulic A, Hudson CC, Homme JL, Yin P, Otterness DM,
Karnitz LM & Abraham RT (2000). A direct linkage between
the phosphoinositide 3-kinase–AKT signaling pathway and
the mammalian target of rapamycin in mitogen-stimulated
and transformed cells. Cancer Res 60, 3504–3513.
Shah OJ, Anthony JC, Kimball SR & Jefferson LS (2000).
4E-BP1 and S6K1: translational integration sites for
nutritional and hormonal information in muscle.
Am J Physiol Endocrinol Metab 279, E715–E729.
Stolovich M, Tang H, Hornstein E, Levy G, Cohen R, Bae SS,
Birnbaum MJ & Meyuhas O (2002). Transduction of growth
or mitogenic signals into translational activation of TOP
mRNAs is fully reliant on the phosphatidylinositol 3-kinasemediated pathway but requires neither S6K1 nor rpS6
phosphorylation. Mol Cell Biol 22, 8101–8113.
Tamaki T, Uchiyama S, Uchiyama Y, Akatsuka A, Yoshimura S,
Roy RR & Edgerton VR (2000). Limited myogenic response
to a single bout of weight-lifting exercise in old rats.
Am J Physiol Cell Physiol 278, C1143–C1152.
Terada N, Patel HR, Takase K, Kohno K, Nairn AC & Gelfand
EW (1994). Rapamycin selectively inhibits translation of
mRNAs encoding elongation factors and ribosomal proteins.
Proc Natl Acad Sci U S A 91, 11477–11481.
Thomson DM & Gordon SE (2005). Diminished overloadinduced hypertrophy in aged fast-twitch skeletal muscle is
associated with AMPK hyperphosphorylation. J Appl Physiol
98, 557–564.
C 2006 The Authors. Journal compilation C 2006 The Physiological Society
305
Wang X, Beugnet A, Murakami M, Yamanaka S & Proud CG
(2005). Distinct signaling events downstream of mTOR
cooperate to mediate the effects of amino acids and insulin
on initiation factor 4E-binding proteins. Mol Cell Biol 25,
2558–2572.
Wang X, Li W, Williams M, Terada N, Alessi DR & Proud CG
(2001). Regulation of elongation factor 2 kinase by p90
(RSK1) and p70, S6 kinase. EMBO J 20, 4370–4379.
Welle S (2002). Cellular and molecular basis of age-related
sarcopenia. Can J Appl Physiol 27, 19–41.
Welle S, Bhatt K & Thornton C (1996a). Polyadenylated RNA,
actin mRNA, and myosin heavy chain mRNA in young and
old human skeletal muscle. Am J Physiol 270, E224–E229.
Welle S, Thornton C & Statt M (1995). Myofibrillar protein
synthesis in young and old human subjects after three
months of resistance training. Am J Physiol 268, E422–E427.
Welle S, Totterman S & Thornton C (1996b). Effect of age on
muscle hypertrophy induced by resistance training.
J Gerontol A Biol Sci Med Sci 51, M270–M275.
Weng QP, Kozlowski M, Belham C, Zhang A, Comb MJ &
Avruch J (1998). Regulation of the p70, S6 kinase by
phosphorylation in vivo. Analysis using site-specific antiphosphopeptide antibodies. J Biol Chem 273, 16621–16629.
Wong TS & Booth FW (1990a). Protein metabolism in rat
gastrocnemius muscle after stimulated chronic concentric
exercise. J Appl Physiol 69, 1709–1717.
Wong TS & Booth FW (1990b). Protein metabolism in rat
tibialis anterior muscle after stimulated chronic eccentric
exercise. J Appl Physiol 69, 1718–1724.
Yarasheski KE, Zachwieja JJ & Bier DM (1993). Acute effects of
resistance exercise on muscle protein synthesis rate in young
and elderly men and women. Am J Physiol 265, E210–E214.
Acknowledgements
We gratefully thank Elizabeth Hedberg for technical assistance
on certain aspects of this project. This research was supported by
an American College of Sports Medicine Foundation Doctoral
Student Research Grant (D.M.T.) and NIH AG025101 (S.E.G.).